CN1067392C - Stable crystalline tetrahydrofolic acid salts - Google Patents

Abstract

The present invention relates to stable crystalline salts of (6R,S)-, (6S)- and (6R)-tetrahydrofolate, to processes for the preparation of these salts and to their use for the production of medicaments and pharmaceutical preparations.

Description

Stable crystalline tetrahydrofolate

The present invention relates to crystalline N-[4-[[2-amino-1,4,5,6,7,8-hexahydro-4-oxo-(6S)-, -(6R)- and -(6R , S)-pteridyl)methyl]amino]benzoyl]-L-glutamic acid salts (hereinafter referred to as tetrahydrofolates), their use and their methods of preparation.

Tetrahydrofolate derivatives contain two asymmetric centers. In this regard, since these derivatives are synthesized from folic acid, N-(pteroyl)-L-glutamic acid, the optically active carbon atom contained in the glutamic acid group exists in the L-form, while the pteroyl 5 , the hydrogenation of the double bond in the 6-position exists in the form of a racemic (6R,S) optically active carbon atom in the 6-position. The synthetic derivative of tetrahydrofolate thus consists of a 1:1 mixture of the 2 diastereomers.

As a drug, tetrahydrofolate mainly uses the calcium salt of 5-formyl-5,6,7,8-tetrahydrofolate (leucovorin) or 5-methyl-5,6,7,8- Calcium salt of tetrahydrofolate, used in the treatment of macrocytic folic acid panemia, as an antidote to improve the compatibility of folic acid antagonists, especially aminopterin and methotrexate, in cancer therapy ("Antifolate rescue ”), for enhancing the therapeutic effect of fluorinated pyrimazoles and for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis, for enhancing the efficacy of certain parasiticides such as trimethoprim-sulfamethoxazole compatibility and for reducing the toxicity of didazatetrahydrofalates in chemotherapy. In humans, individual tetrahydrofolates are interconvertible (folate cycle). Tetrahydrofolate plays a major role in such a process. Tetrahydrofolate is also used as a base material for the preparation of various tetrahydrofolate derivatives.

So far, due to the difficulty of producing tetrahydrofolate with qualified purity as the active ingredient of the drug and the extreme instability of tetrahydrofolate, especially its high sensitivity to oxidation, tetrahydrofolate is directly used as a drug or used to prepare various All base materials for tetrahydrofolate derivatives failed [on this point see A.L. Fitzhugh, Pteridine 4(4), 187-191 (1993)]. In this regard, it should be pointed out that for the parenteral application of tetrahydrofolate, which is the most commonly used in the pharmaceutical field, the solution administered is at least close to neutral pH as a prerequisite that must be followed. For this application, tetrahydrofolate is therefore the preferred application form. In order to overcome the instability of tetrahydrofolate, various methods have been employed, such as excluding oxygen as completely as possible, or adding an antioxidant such as ascorbic acid. However, for the use of medicines, it is almost impossible to completely eliminate oxygen, and even if it is possible, it will require a high cost; it is also not possible to add antioxidants for pharmaceutical applications. Therefore, so far, no feasible method suitable for commercially preparing tetrahydrofolate with high purity and sufficient stability to make the pharmaceutical application of tetrahydrofolate possible has not been found.

Surprisingly, it has now been found that (6S)- or (6R)-enriched tetrahydrofolate can be salt, or (6R,S)tetrahydrofolate crystallization, to obtain high-purity (6S)-, (6R)- or (6R,S)-tetrahydrofolate with excellent stability. The crystalline (6S)-, (6R)- and/or (6R,S)-tetrahydrofolate salts thus obtained are practically extremely stable in suitable form at room temperature. They are suitable as components of medicines or raw materials for the production of medicines, or as raw materials for the industrial production of other tetrahydrofolate derivatives with high purity.

Accordingly, the present invention relates to crystalline salts of (6R,S)-, (6S)- and (6R)-tetrahydrofolate. Alkaline earth metal salts, especially calcium or magnesium salts, are preferably used as crystalline tetrahydrofolate.

The invention also relates to a process for the preparation of crystalline (6R,S)-, (6S)- and (6R)-tetrahydrofolates, characterized in that the corresponding tetrahydrofolates are crystallized. In this respect tetrahydrofolate is preferably crystallized from a polar medium at a pH of 7-10.

The most suitable substance as a polar medium is water or a mixture of water-miscible organic solvents, such as water-soluble alcohols such as methanol, ethanol, n-propanol, isopropanol, ethylene glycol, low molecular weight aliphatic water-soluble carboxylic acids Such as formic acid, acetic acid or lactic acid, or water-soluble amides such as formamide, dimethylformamide, dimethylacetamide, 1-methylpyrrolidone, 2-methylpyrrolidone or 2-piperidone. There are no specific restrictions on the type of solvent used and the mixing ratio, since crystalline tetrahydrofolate generally has lower solubility than the corresponding amorphous form.

Crystallization is preferably carried out at elevated temperature, especially at 50-90°C, or crystallization from dilute solution, especially 1-10% solution.

Crystallization of (6S)-, (6R)- and (6R,S)-tetrahydrofolates occurred spontaneously or by inoculation with the corresponding crystalline tetrahydrofolates.

Amorphous or crystalline pure (6S)- or (6R)-tetrahydrofolate is preferably used as starting material for crystallization. However, racemic (6R,S)-tetrahydrofolate can also be used, since (6S)- or (6R)-tetrahydrofolate can be enriched. Suitable starting materials here include two isolated solids such as (6R,S)-tetrahydrofolate, (6S)-tetrahydrofolate-sulfuric acid and sulfonic acid addition salts prepared according to EP-495204, and by catalytic hydrogenation or borohydride reduction of tetrahydrofolate prepared in situ from folic acid.

Since amorphous or partially crystalline optically pure tetrahydrofolate or its salts are used as starting material for crystallization, it is possible to obtain previously unattainable purity (>98%) and stability of the salt with the described method.

The present invention also relates to the use of crystalline (6S)-, (6R)- and/or (6R,S)-tetrahydrofolate as an ingredient in the production of medicaments or for the preparation of other tetrahydrofolate derivatives, because the crystalline ( 6S)-, (6R)- and (6R,S)-tetrahydrofolates retain a very good quality, due to their excellent stability in solid form, virtually unchanged for an unlimited time. The invention also relates to pharmaceutical preparations containing crystalline (6S)-, (6R)- and/or (6R,S)-tetrahydrofolate. The pharmaceutical preparations can be produced by known methods, such as freeze-drying. The solubility of crystalline tetrahydrofolate in water at 20°C is less than 1 mg/ml. Used similarly to substances known in the art for tetrahydrofolate (eg 5-formyl-5,6,7,8-tetrahydrofolate).

Furthermore, the present invention relates to a method for the separation of the magnesium salt of (6R,S)-tetrahydrofolate into two diastereoisomers, namely (6S)- and (6R)-tetrahydrofolate, by fractional crystallization of magnesium salts. The method is simple and effective. Even after the first crystallization of the crude racemic (6R,S)-magnesium tetrahydrofolate salt, more than 95% of the (6R)-molecular The yield of the body is greater than 50%. Crystalline (6S)- and (6R)-tetrahydrofolate magnesium salts can be obtained in higher isomeric purity by further crystallization under similar conditions.

In each case, the contents of tetrahydrofolate and isomeric fractions given in the examples were determined by HPLC.

Embodiment 19 (stability)

To determine the stability of crystalline (6S)- and (6R)-tetrahydrofolate, these substances were stored together with a control at 25°C, 60% relative temperature, under nitrogen or under air at 4°C. The remaining THF content was measured at certain time intervals and compared with the initial value. Exposure time, months 0 1/2 1 2 6 9 Crystalline (6S)-Tetraargon 25°C, 60%, N ₂ calcium salt of folic acid 4°C, air 100%100% 98.5%95.5% 97.2%92.4% 96.8%89.2% 97.7%82.3% 97.7%76.0% Crystalline (6R)-tetraargon 25°C, 60%, N ₂ Calcium salt of folic acid 4°C, air 100%100% 97.8%85.9% 96.1%79.0% 98.0%73.0% 97.1%69.3% 96.8%69.2% Crystalline (6R,S)-Tetraargon 25°C, 60%, N ₂ calcium salt of folic acid 4°C, air 100%100% 99.3%96.5% 98.0%94.2% 99.0%89.0% 97.8%86.6% 98.7%86.2%

After prolonged storage, crystalline tetrahydrofolate retains a very light color. In contrast, amorphous samples rapidly and strongly discolored.

After exposure to air at 4°C for 1 month, the content of amorphous (6R,S)-tetrahydrofolate calcium salt was 8% lower than that of crystalline (6R,S)-tetrahydrofolate calcium salt.

Embodiment 2 (X-ray powder diffraction spectrum)

In order to characterize the structural properties (degree of crystallinity) of the crystalline tetrahydrofolate salts, the X-ray powder diffraction spectra of these substances were determined.

Crystalline (6S)-, (6R)- and (6R,S)-tetrahydrofolate gave good resolution, narrow bands and low background spectra. The pattern shows a high crystalline content.

Example 3 [TE1423]

Slurry 8.2 g of (6R,S)-tetrahydrofolate in 100 ml of water containing 1 g of thioglycerol under nitrogen, adjust the pH to 3.3 with 30% aqueous sodium hydroxide solution, then dissolve with 3.8 g of calcium chloride in 4 g of water solution for treating slurries. The resulting solution had a pH of 9.3. After stirring at room temperature for 20 h, the resulting suspension had a pH of 10.0, which was filtered under suction and the residue was washed with a little water.

After drying, 7.7 g of reddish crystalline (6R,S)-tetrahydrofolate calcium salt was obtained, the (6S) fraction was 51.1%, and the content was 96%.

Example [TE1418]

28.6 g (6R,S)-tetrahydrofolate was slurried in 114 ml water under nitrogen, the pH was adjusted to 7.5 with 30% aqueous sodium hydroxide solution, and the slurry was treated with a solution of 48.5 g calcium chloride in 500 ml water . The resulting rubber-like mass was heated to 90°C. After stirring for 1 h, a bright yellow suspension was obtained, which was filtered hot under suction and washed with a little water.

After drying, 17.3 g of beige crystalline calcium salt of (6R,S)-tetrahydrofolate was obtained, the (6S) fraction was 50.9%, and the content was 94%.

Example 5 [Am593]

Slurry 4.0 g of (6R,S)-tetrahydrofolate in 40 ml of water containing 0.4 g of thioglycerol under nitrogen, adjust the pH to 8.5 with 30% aqueous sodium hydroxide, and then add 2.0 g of calcium acetate at 50 °C Next process the slurry. The beige product which slowly crystallized from the resulting solution was filtered under suction, and the residue was washed with water.

After drying, 3.64 g of crystalline (6R,S)-tetrahydrofolate calcium salt were obtained with a (6S) fraction of 50.5% and a content of 94.6% (as a salt, relative to the dry substance). The calcium content is 1.12 equivalents.

Example 6 [Am592]

12.0 g (6S)-tetrahydrofolate was slurried in 60 ml of water containing 0.6 g of thioglycerol under nitrogen, the pH value was adjusted to 7.5 with 50% aqueous sodium hydroxide solution, and then 22.5 g of calcium chloride was added to 20 ml of water. Solution Process the slurry at 85°C. After stirring at 85° C. for 2 h, the crystallized product was filtered under suction and washed with water.

After drying, 12.9 g of crystalline calcium salt of (6S)-tetrahydrofolate was obtained, with a (6S) fraction of 99.9% and a content of 96.8%. The product thus obtained has a solubility in water of 0.12% at pH 6 at 50°C.

Example 7 [Am602]

Using 12.0 g of (6R)-tetrahydrofolate and a treatment similar to that described in Example 6, 13.8 g of crystalline (6R)-tetrahydrofolate calcium salt was obtained with a (6R) fraction of 99.0% and a content of 93%. The product thus obtained has a solubility in water of 0.07% at pH 6 at 50°C.

Example 8 [Am482]

Slurry 40.0 g of (6S)-tetrahydrofolic acid in 160 ml of water under nitrogen, and adjust the pH value to 9.8 with 25% ammonia water at 0-5°C. A solution of 34 g of magnesium chloride in 34 ml of water was added to the resulting solution. After adjusting the pH to 7.0 and adding 200 ml of ethanol, the off-white product which crystallized out was filtered off with suction and washed with ethanol/water.

After drying, 37.0 g of crystalline magnesium salt of (6S)-tetrahydrofolate was obtained, the (6S) fraction was 99.4%, and the content was 91.7%.

Example 9 [Am583]

40.0 g of (6R)-tetrahydrofolate was slurried in 400 ml of water containing 4 g of thioglycerol under nitrogen, treated with 6.0 g of magnesium hydroxide and 60.0 g of magnesium acetate. Adjust the pH to 9.0 with 25% aqueous ammonia at 50°C. After cooling to 20°C, a gummy mass was obtained which was transferred to a fluid suspension with heating to 35°C. The suspension was filtered under suction at 35° C. and washed with water.

After drying, 18.0 g of crystalline magnesium salt of (6R)-tetrahydrofolate was obtained, with a (6R) fraction of 99.4% and a content of 92.0%.

Example 10 [Am590]

20.0 g of (6R,S)-tetrahydrofolate were slurried in 200 ml of water containing 2 g of thioglycerol under nitrogen, treated with 2.7 g of magnesium hydroxide, and heated to 50°C. After adding 30 g of magnesium acetate, the pH was adjusted to 7.3 with 25% aqueous ammonia, and the solution was cooled to 20° C. and stirred overnight. The resulting suspension was filtered off with suction and washed with water.

After drying, 5.0 g of crystalline magnesium salt of (6R)-tetrahydrofolate was obtained, with a (6R) fraction of 94.8% and a content of 97.1%.

Example 11 [Am506]

Slurry 28.0 g of (6R,S)-tetrahydrofolate in 110 ml of water and 75 ml of methanol containing 18 g of thioglycerol under nitrogen, treat with 9.5 g of magnesium hydroxide, then adjust the pH with 25% aqueous ammonia at 50 °C Adjust to 9.3. The fine suspension obtained after cooling to -5° C. was filtered off with suction and washed with a cold methanol/water mixture.

After drying, 10.5 g of crystalline magnesium salt of (6R,S)-tetrahydrofolate was obtained, with a (6S) fraction of 49.5% and a content of 95.9%.

Example 12 [Am497]

4.0 g of (6R,S)-tetrahydrofolate was slurried in 16 ml of water under nitrogen, the pH was adjusted to 9.7 with 25% aqueous ammonia, and the slurry was treated with a solution of 3.2 g of magnesium chloride in 3.2 ml of water. The resulting clear solution was added to 200 ml of ethanol. After stirring for 2 h, the resulting pale yellow suspension was filtered under suction at 5° C. and washed with a cold ethanol/water mixture.

After drying, 4.5 g of crystalline magnesium salt of (6R,S)-tetrahydrofolate was obtained, the (6S) portion of which was 49.3%, and the content was 90.3%.

Claims (8)

1. A crystalline calcium salt of (6R,S)-, (6S)- and (6R)-tetrahydrofolate. 2. A crystalline magnesium salt of (6R,S)-, (6S)- and (6R)-tetrahydrofolate. 3. A method for preparing crystalline (6R, S)-, (6S)- and/or (6R)-calcium or magnesium salts of tetrahydrofolate, characterized in that (6R, S)-, (6R)- Or (6S)-tetrahydrofolate crystallizes in polar medium at pH 7-10. 4. The method according to claim 3, characterized in that the crystallization is carried out at elevated temperature and/or from a dilute solution. 5. Process according to claim 4, characterized in that the crystallization is carried out in water or in a mixture of water-miscible organic solvents. 6. Process according to claim 5, characterized in that acetic acid or a low molecular weight organic alcohol is used as water-miscible solvent. 7. Use of calcium or magnesium salts of crystalline (6R,S)-, (6S)- or (6R)-tetrahydrofolate as ingredients for the manufacture of medicaments. 8. Pharmaceutical preparations containing calcium or magnesium salts of crystalline (6R,S)-, (6S)- or (6R)-tetrahydrofolate.